Method of preparing tetanus toxin



- with hot air prior to its introduction into the Patented Apr. 10, 1945 I I 2,373,454

UNITED [STATES PATENT OFFICE METHOD OF PREPARING TETANUS roxm William Edward Bunney, Millstone, and Walter Ludwig Koerber, Milltown, N. .L, assignors to E. R. Squibb & Sons, New York, N. Y., a corporation of New York No Drawing. Application July 9, 1943.

Serial No. 494,076

. 7 Claims. ('01. 167-78) This invention relates to the preparation of The following examples are illustrative of the agents for the prophylaxis and therapy of invention: tetanus. Example 1 Tetanus, or lockjaw, is an infectious disease caused by certain anaerobic bacteria. Immuni- 5 zation against this disease is efiected with tetanus toxoid, which is the non-toxic product obtained by incubating Clostridium te'tani in a suitable medium, separating the tetanus toxin formed, and treating the toxin with formaldehyde; and from the serum of horses immunized by repeated injections of tetanus toxin,- an antitox-in is obtained which is useful in both the prophylaxis and the therapy of tetanus.

It is an object of this invention to provide a It! method of preparing improved tetanus toxins; and, further, a method of preparing improved (a) Activated charcoal (e. g., Nuchar, C-1l 5, neutral) is sterilized twice with air heated to weight to a peptone b'roth lcf. "A compilation organisms" by'Levine and Schoenlein, published by Williams and Wilkins 00., Baltimore, 1930 No. 748, page 214]; and the charcoal-containing medium is sterilized for minutes at'15 1b. pressure in 3-liter Erlenmeyer flasks containing 2700 ml. medium. After cooling, the medium is seeded with a-selected strain of Clostridium tetani and incubatedfor 10-12 days at 35 'C.

tetanus toxoids.

- I (The charcoal stays suspended throughout the In 1917 colgbmok medium during the wholeincubatlon-period and (Lancet II, 530-532) observed that a mediumto 20 v 1 e gives the flasks an opaque appearance.) ..At the WhlCh activated charcoal had been added was d f th incubati fled charcoal and rendered suitable for the growth of anaerobic g e re g g fflltra'uon through bacilli without any further precautions as to O g sms y anaeric conditions; and they alsofnoted that more rapid growth of Clostrz'dium 'tetam' was obtained in liquid media containing charcoal.

It has now been found that the presence of activated charcoal (active carbon) in peptone-containing media during the growth of 'C'lostridium tetam' not only influences its growth, but also favorably influences the combining power (L-Hmi.) of its most important metabolite, the tetanus toxin. Thus; toxins 1 prepared in this manner show exceptionally high values of combining power (and the toxoids obtained therefrom show increased antigenicity) as compared with control toxins prepared in the absence of activated charcoal. The desirability of higher antigenicity in tetanus toxoids is obvious;

- In general, the method of this invention comprises incubating Clostridz'um tetani in a liquid peptone-containing medium in the presence of a small proportion (say, about 0.1-1.0%- by weight) of activated charcoal, and separating the toxin from the charcoal and organisms. The optimal proportion of charcoal is about 0.2% by weight. Preferably, the charcoal issterilized Samples of the thus-obtained toxin'in peptone diluent, when tested in guinea pigs; exhibit a combining power about twice that of a control toxin prepared without the use of activated charcoal.

(a) The toxin obtained as described in (a) is converted into a' toxoid by adding 02-03% formalin, incubating at 37 C. for three weeks, treating the toxoid solution with a sterile solutained, and resuspending the precipitate in toxoid thus obtained have about twice the -antigenic'power of a provisional standard tetanus toxoid, as compared with a control toxoid (made from a' toxin simultaneously prepared on the identical medium and with the same strain of organism, but without charcoal), which has about the same antigenic power as the standard.

removal of colored matter from the medium by the charcoal. Example 2 medium. Preferably also, the pressure above Theprocedure of Example 1 (a) is followed, the medium is continuously or periodically re- 50 except that the medium is replaced by Martin's duced during the incubation period. stomach-digest infusion broth (of. No. 1006, page 160-180 C., and added to the extent-of 0.2% by of culture media for the cultivation of. micro-- (Monographs on systematic bacteriology, v. 2)

paper pulp pads or by centrifugation, followed by filtration through 13-1b. Berkefeld candles.

' tion of alum until complete precipitation is obphysiological salt solution. Samples of the Moreover, the toxoid is colorless, owing to the,

Example 3 The procedure of Example 1(a) is followed,

except that the medium is replaced by' Martin's stomach-digest infusion broth, the flasks are placed .in a vacuum chamber in an incubator kept at 35 C., and the pressure above-the medium is reduced twice daily to about 100-200 mm. Samples of the toxin thus obtained exhibit about four times the combining power of the corresponding control (with no charcoal and no evacuation). The toxoid obtained from this toxin, following the procedure of Example 1(b),

has a correspondingly high antigenic power.

The improved toxins obtained in accordance with the foregoing examples may be used advantageously forthe production of antitoxins; thus, an eflicacious antitoxin may be obtained (in the conventional manner) by refining and concentrating the serum of horses immunized by repeated injections of these improved tetanus toxins (after a preliminary injection of a-tetinus toxoid) Also, the method of this invention may be employed in the preparation of combined tetanus and gas-gangrene antitoxin; e. g., by immunizing horses with gas-gangrene toxins and the improved tetanus toxin of this invention. Furthermore, the improved toxoid of this invention may be used for the preparation of a combined liquid or alum precipitated tetanus perfringens (Welchii) toxoid for human used Other activated charcoals (active carbons) may be employed instead of Nuchar in the procedure of the foregoing examples, inter alia, Dame and Norite.

The invention may be'variously otherwise embodied within the scope of the appended claims.

We claim:

i. The method of preparing a tetanus toxin which-comprises incubating ,Clostridium tetam' in aliquid peptone-containing medium in the presence of a small proportion of activated charcoal,

and separating the toxin from the charcoal and organisms.

2. The method of preparing a tetanus toxin which comprises incubating Clostridium tetani in a liquidpeptone-containing medium in the presence of a small proportion of a hot-air-steriiized activated charcoal, and separating the toxin from the charcoal and organisms.

3. The method of preparing a tetanus toxin which comprises incubating Clostridium tetani in a liquid peptone-containing medium in the presence of about 0.1-1.0% by weight of activated charcoal, and separating the toxin from the charcoal and organisms.

4. The method-ct preparing a tetanus toxin which comprises incubating C'lostridium tetani in a liquid peptone-containing medium in the presence of about 0.2% by weight of activated charcoal, and separating the toxin from the charcoal and organisms.

5. The method of preparing a tetanus toxin which comprises incubatingClostridium tetani in a peptone broth in the presence of a small proportion of activated-charcoal, and separating the toxin from the charcoal and organisms.

6. The method of preparing a tetanus toxin I which comprises incubating Clostridz'um tetani .WILLIAM EDWARD BUNNEY. WALTER L. KOERBER. 

